Powder containing physiologically active peptide

ABSTRACT

A method is disclosed for stabilizing a physiologically active peptide in a process of preparing a powder containing the physiologically active peptide by drying an aqueous liquid containing the physiologically active peptide, wherein the method comprises adding to the aqueous liquid at least one compound selected from the group consisting of a nonionic surfactant, a water-soluble, nonionic, organic binder, hydrogenated lecithin, and mannitol.

FIELD OF THE INVENTION

[0001] The present invention relates to a physiologically activepeptide-containing powder, and in particular to a physiologically activepeptide-containing powder in which contamination by denatured peptideshas been suppressed by stabilizing the physiologically active peptideand thereby preventing its denaturation from taking place in the processof forming a powder by drying an aqueous liquid containing thephysiologically active peptide. The present invention further relates toa physiologically active peptide-containing powder suitable fortranspulmonary and transnasal administration by inhalation.

BACKGROUND OF THE INVENTION

[0002] Administration of pharmaceutical products containing aphysiologically active peptide have been made, so far, by injection. Inthis context, lyophilization has exclusively been employed in thepreparation of such pharmaceutical compositions. Thus, for suchpharmaceutical compositions, studies addressed to the stabilization oftheir active components, physiologically active peptides, have so farbeen focused on either the long-term storage stability of thephysiologically active peptides in a dry state pharmaceuticalcompositions of the final products, or the storage stability of thephysiologically active peptides in liquids which are prepared bydissolving the peptide-containing dry compositions. For example,stabilization of calcitonin solutions is disclosed in JapaneseUnexamined Patent Publication Nos. H07-179364, H07-188060 andH07-188061, and stabilization of lyophilized growth hormone products isdisclosed in Japanese Unexamined Patent Publication Nos. H10-504531,H10-511965 and H10-507183.

[0003] The reason why injection has been the sole way for administeringphysiologically active peptides is that, when they are orallyadministered, physiologically active peptides are digested in thegastrointestinal tract. A practically applicable new route foradministration, if established, would provide a great benefit topatients. Above all, in the case of active peptides requiring lifelongadministration such as growth hormone and insulin, the conventional wayof administration of injection has been giving patients inconvenienceand pain. For these physiologically active peptides, therefore,establishment of a route of administration other than injection has beenlonged for by the patients.

[0004] On the other hand, those pharmaceutical compositions for systemicadministration of a drug are under investigation that are intendedeither for transpulmonary absorption of a pharmacologically activeingredient by inhalation (referred to as an “inhalant composition” inthe present specification) or for absorption of such an ingredientthrough the nasal mucous membrane by intranasal application, i.e.compositions for transnasal administration, as compositions utilizingother, new administration routes than those relied on by conventionalpharmaceutical compositions such as injections, oral preparations,suppositories and the like. Inhalant compositions and compositions fortransnasal administration are not directly injected into the body, butthey are applied onto the surface of mucous membranes which are exposedto the air such as membranes of the respiratory tract. Therefore, theirstandards for microbiological quality control are not so strict as thosefor injections. Thus, they may be produced not only by a lyophilizationapparatus but also by a fluid-bed granulation apparatus, a spray dryingapparatus, or a spray-freeze drying apparatus. Concerning stabilizationof active peptides in production steps of pharmaceutical compositionsusing a fluid-bed granulation apparatus, a spray drying apparatus, or aspray-freeze drying apparatus, it is reported that stabilization isattained by addition of an inhibitor of Maillard reaction (JapaneseUnexamined Patent Publication No. H10-505591). However, it ispreferable, if possible, that stabilization of a given active peptide ina production process should be achieved by means of approvedpharmaceutical additives which are highly safe and have been used foryears. This is because such an additive would allow to expect highersafety with regard to the final pharmaceutical product obtained. It isalso required that the absorption and transferal to the blood of anphysiologically active peptide is attained in sufficient efficiency.

[0005] The present invention has as its objectives to provide a methodto improve stability of a physiologically active peptide in a process ofproducing a powder by drying an aqueous liquid containing thephysiologically active peptide, as well as to provide a physiologicallyactive peptide-containing powder produced by the method.

[0006] The present invention has as its further objectives to provide aphysiologically active peptide-containing powder especially suited forabsorption of the physiologically active peptide by inhalation, and toprovide an inhalant composition.

SUMMARY OF THE INVENTION

[0007] For production of a powder containing a physiologically activepeptide, the present inventors found that, in a process of preparing apowder containing a physiologically active peptide by drying an aqueousliquid containing the peptide, addition of certain compounds to theaqueous liquid remarkably increases the stability of the physiologicallyactive peptides during the powder preparation. In addition, the presentinventors also found that physiologically active peptides contained inthe powder thus prepared are efficiently absorbed into the blood whenthe powder is transpulmonarily administered. The present invention wasmade on the basis of these findings.

[0008] Thus, the present invention provides a method for stabilizationof a physiologically active peptide in a process of preparing a powdercontaining the physiologically active peptide by drying an aqueousliquid containing the physiologically active peptide, wherein the methodcomprises adding to the aqueous liquid at least one compound selectedfrom the group consisting of a nonionic surfactant, a water-soluble,nonionic, organic binder, hydrogenated lecithin, and mannitol. In themethod, a nonionic surfactant, a water-soluble, nonionic, organicbinder, hydrogenated lecithin and mannitol serve as stabilizers inpreparing a powder containing a physiologically active peptide from anaqueous liquid containing it. Thus, one or more of these compoundsemployed suppress denaturation such as dimer formation in the process offorming a powder from an aqueous liquid containing the peptide, therebyenabling to prepare a physiologically active peptide-containing powderwhich is substantially free of denatured peptides.

[0009] The present invention further provides a method for stabilizationof a physiologically active peptide in a process of preparing a powdercontaining the physiologically active peptide by drying an aqueousliquid containing the physiologically active peptide, wherein the methodcomprises adding to the aqueous liquid mannitol and at least onecompound selected from the group consisting of a nonionic surfactant, awater-soluble, nonionic, organic binder, and hydrogenated lecithin. Thismethod enables, in addition to the above-mentioned benefit, to prepare apowder effecting especially efficient transpulmonary absorption of aphysiologically active peptide.

[0010] In the above methods for stabilization, with regard to a nonionicsurfactant or a water-soluble, nonionic, organic binder added to theaqueous liquid, the concentration range where they exhibit a potentstabilizing effect is 0.01-0.5 % by weight for a nonionic surfactant and0.01-1 % by weight for a water-soluble, nonionic, organic binder. As formannitol, it exhibits a potent stabilizing effect when added in anamount of 1-50 parts by weight per one part by weight of aphysiologically active peptide.

[0011] In the above, it is more preferable that the nonionic surfactantis selected from the group consisting of polysorbate,polyoxyethylenehydrogenated castor oil, and a poloxamer (polyoxyethylenepolyoxypropylene block copolymer: Pluronic).

[0012] Also in the above, the water-soluble, nonionic, organic binder ismore preferably selected from the group consisting ofpolyvinylpyrrolidone, a water-soluble, nonionic, cellulose derivativeand polyvinylalcohol.

[0013] Further, the water-soluble, nonionic, cellulose derivative ismore preferably selected from the group consisting ofhydroxypropylcellulose, hydroxyethylcellulose, andhydroxypropylmethylcellulose.

[0014] The effects of these stabilizers are remarkable in the aboverange, though they still have substantial effects somewhat outside theranges. A still more preferable concentration range for a nonionicsurfactant is 0.05-0.3 % by weight, where a particularly potentstabilization effect is obtained. For a water-soluble, nonionic, organicbinder, a concentration range still more preferable than the above is0.02-0.5 % by weight, where a particularly potent stabilization effectis obtained. As for hydrogenated lecithin, its stabilizing effect isparticularly remarkable even at a concentration as low as 0.01% byweight. While its effect peaks at concentrations of 0.5-1 % by weight,the effect remains still remarkable outside this range, and even at 2%by weight. Thus, the decline in its stabilizing effect is only limitedeven when its concentration goes up beyond the peak concentration. Anupper limit concentration, therefore, is not clear over whichhydrogenated lecithin would substantially lose its stabilizing effect.Its concentration, however, may be chosen as desired considering ease ofhandling in production of the pharmaceutical composition as there is noreason for using an unnecessarily large amount of hydrogenated lecithininsofar as it exhibits a sufficient effect as an additive. In general,the concentration of hydrogenated lecithin is preferably in the range ofabout 0.005-4% by weight, and more preferably in the range of 0.01-2% byweight. In light that the total amount of the powder administered is tobe small insofar as it does not prevents easy handling, the weightproportion of a physiologically active peptide to mannitol is morepreferably 1:1 to 1:40, further more preferably 1:1 to 1:30, still morepreferably 1:1 to 1:20, and most preferably 1:1 to 1:10. Forstabilization of an physiologically active peptide, any of the abovestabilizers may be used alone, or two or more of them may be used incombination. When used in combination, they exhibit a still moreremarkable stabilizing effect than when one of them is used alone, thusallowing to almost completely prevent the formation of denatured peptidesuch as a dimer.

[0015] The present invention is characterized in that its uses, indrying an aqueous liquid containing a physiologically active peptide, acertain group of compounds that were found to stabilize active peptides.The compounds can be used in a wide variety of specific methods fordrying. In the above, example of methods for drying aqueous liquidsinclude, but are not limited to, spray drying, lyophilization andspray-freeze drying, and, furthermore, a variety of methods whichinclude a process of drying a solution by spraying it, such as dryingperformed in fluid-bed granulation, in a variety of coating method suchas fluid-bed coating which allow to coat the surface of core particles,as well as drying performed in a granulation process in fluid-bedgranulation involving coating of, or attachment of materials to, thesurface of core particles.

[0016] Inhaled particles are more easily carried on the air flow deepinto the respiratory system when their average size is 1-10 μm, and morepreferably 2-5 μm. When given such a size, the particles of thephysiologically active peptide-containing powder obtained in a stableform by one of the above methods are easily carried deep into therespiratory system by inhalation, allowing efficient and relativelylong-lasting transferal of the physiologically active peptide into thecirculating blood. Thus, the present invention further provides one ofthe above method in which the average size of the particles making upthe powder is 1-10 μm, and more preferably 2-5 μm.

[0017] Examples of active peptides stabilized according to the presentinvention include calcitonins, insulins, growth hormones,erythropoietin, glucagon, somatostatin, somatostatin derivatives,interferons (α, β or γ), interleukins (I, II, III, IV, V VI or VII),superoxide dismutase, urokinase, proteases, tumor necrosis factors,colony-stimulating factors, kallikrein, lysozyme, fibronectin, as wellas a variety of factors regulating growth or differentiation of cellssuch as insulin-like growth factors, epidermal growth factor, fibroblastgrowth factors, platelet-derived growth factor, nerve growth factor,hepatocyte growth factor, vasculogenesis factors, andanti-vasculogenesis factors. As active peptides share a common chemicalstructure that they consist of two or more amino acids linked by peptidebonds, the present invention is also applicable to a wide variety ofother active peptides than those enumerated above. Moreover, it does notmatter whether those peptides have been obtained by extraction fromnatural sauces or produced by application of genetic recombinationtechnology, for such difference will not influence the basicphysicochemical characters of the peptides. Among the above peptides,human growth hormone and human insulin are particularly preferredpeptides in the present invention, for they are such peptides thatpatients have had to continue administering themselves by subcutaneousinjection for a long period of time

[0018] In addition to the above stabilizing methods, the presentinvention provides a method for preparation of a powder containing aphysiologically active peptide. The method for preparation comprisesforming a powder by drying an aqueous liquid containing aphysiologically active peptide and a nonionic surfactant, awater-soluble, nonionic, organic binder, hydrogenated lecithin, and/ormannitol. By one or more of those stabilizers added to a physiologicallyactive peptide, denaturation such as dimer formation is suppressed whilethe physiologically active peptide is in the process of forming a powderfrom an aqueous liquid containing the peptide. Thus, a physiologicallyactive peptide-containing powder is prepared which is substantially freeof denatured peptides.

[0019] The present invention further provides a method for preparationof a powder containing a physiologically active peptide, wherein themethod comprises forming a powder by drying an aqueous liquid containingthe physiologically active peptide, mannitol, and at least one compoundselected from the group consisting of a nonionic surfactant, awater-soluble, nonionic, organic binder, and hydrogenated lecithin. Thismethod for preparation, in addition to the above-mentioned benefit,provides a powder that effects especially efficient transpulmonaryabsorption of a physiologically active peptide.

[0020] In the method for preparation above, the nonionic surfactant ismore preferably selected from the group consisting of polysorbate,polyoxyethylenehydrogenated castor oil, and a poloxamer (polyoxyethylenepolyoxypropylene block copolymer: Pluronic). The water-soluble,nonionic, organic binder is more preferably selected from the groupconsisting of polyvinylpyrrolidone, a water-soluble, nonionic, cellulosederivative and polyvinylalcohol. The water-soluble, nonionic, cellulosederivative is more preferably selected from the group consisting ofhydroxypropylcellulose, hydroxyethylcellulose, andhydroxypropylmethyl-cellulose.

[0021] In the method for preparation above, preferable ranges of theamount of the enumerated stabilizers when employed are the same as thosementioned for them in the method for stabilization of physiologicallyactive peptides above. Therefore, a still more preferable concentrationrange for a nonionic surfactant is 0.05-0.3% by weight, and, for awater-soluble, nonionic, organic binder, a still more preferableconcentration range is 0.02-0.5% by weight. As for hydrogenatedlecithin, an upper limit concentration is not clear over whichhydrogenated lecithin would substantially lose its stabilizing effect.Its concentration, however, may be chosen as desired considering ease ofhandling in production of the pharmaceutical composition as there is noreason for using an unnecessarily large amount of hydrogenated lecithininsofar as it exhibits a sufficient effect as an additive. In general,the concentration of hydrogenated lecithin is preferably in the range ofabout 0.005-4% by weight, and more preferably in the range of 0.01-2% byweight. As to mannitol, the weight proportion of a physiologicallyactive peptide to mannitol is more preferably 1:1 to 1:40, further morepreferably 1:1 to 1:30, still more preferably 1:1 to 1:20, and mostpreferably 1:1 to 1:10.

[0022] In the method for preparation above, example of methods fordrying aqueous liquids include, but are not limited to, spray drying,lyophilization and spray-freeze drying, and fluid-bed granulation, aswell as a variety of coating method, such as fluid-bed coating, whichallow to coat the surface of core particles, and fluid-bed granulationinvolving coating of, or attachment of materials to, the surface of coreparticles.

[0023] In the method for preparation above, the average size of theparticles making up the powder is preferably 1-10 μm, and morepreferably 2-5 μm, when considering transpulmonary administration of aphysiologically active peptide.

[0024] The range of physiologically active peptides formed into a powderby the method for preparation above is the same as already mentionedwith regard to the method for stabilization.

[0025] The present invention further provides a powder containing aphysiologically active peptide, wherein the powder is made up ofparticles comprising a physiologically active peptide and mannitol at aweight proportion of 1:1 to 1:50. In the powder, more preferably, theparticles making up the powder further comprise, per one part by weightof the physiologically active peptide, at least one component selectedfrom the group consisting of a nonionic surfactant in an amount of0.05-3 parts by weight, a water-soluble, nonionic, organic binder in anamount of 0.05-6 parts by weight, and hydrogenated lecithin. Such apowder effects an efficient absorption of a physiologically activepeptide through a mucous membrane deep in the respiratory system.

[0026] Considering reduction of the total weight of the powder inhaledper a predetermined amount of a physiologically active peptide to beadministered, the weight proportion of a physiologically active peptideto mannitol in the particles above is more preferably 1:1 to 1:40,further more preferably 1:1 to 1:30, still more preferably 1:1 to 1:20,and most preferably 1:1 to 1:10. The amount of a nonionic surfactant ismore preferably 0.25-1.8 parts by weight per one part by weight of aphysiologically active peptide, in which range efficient absorption of aphysiologically active peptide is attained while suppressing the amountof a nonionic surfactant employed. Likewise, the amount ofwater-soluble, nonionic, organic binder is more preferably 0.1-3 partsby weight per one part by weight of a physiologically active peptide.

[0027] In the above powder containing a physiologically active peptide,the average size of the particles making up the powder is preferably1-10 μm, and more preferably 2-5 μm. By giving such an average size toits particles, the powder becomes easily carried deep into therespiratory system by inhalation, allowing more efficient absorption ofthe physiologically active peptide.

[0028] Method for preparation of the above powder containing aphysiologically active peptide is not limited. The powder may beprepared, for example, by spray drying, spray-freeze drying orlyophilization.

[0029] The range of physiologically active peptides in the above powdercontaining a physiologically active peptide is the same as alreadymentioned with regard to the method for stabilization.

[0030] The present invention further provides an inhalant compositioncontaining a physiologically active peptide, wherein the inhalantcomposition comprises above-mentioned particles containing aphysiologically active peptide. The inhalant composition may simply besuch particles containing a physiologically active peptide, or they maybe either clusters consisting of such particles loosely associated withone another or composites consisting of such particles plus larger,inert carrier particles (e.g. lactose) onto the surface of which theformer particles are loosely attached. Such loose clusters or compositesare constructed in an extent of looseness that, at the time of inhalingthe composition, they will be disintegrated upon release from aninhalation device by the flow of air and the each fine particlecontaining a physiologically active peptide will thereby be liberatedfrom the clusters or carriers into a separate particle Preparation ofsuch loose clusters or loose and coarse composite particles can beprepared by any of a variety of methods well known to those skilled inthe art for bringing particles of the size of one to several μm makingup a powder into a loose association with one another or into a looseassociation onto larger, inert carrier particles. Such loose clusters orloose and coarse composite particles are intended to increaseflowability of the composition for improved ease of filling and accuracyof filling amount in a process in which a unit dose of the inhalantcompositions is filled into each of predetermined containers likecapsules employed in a inhalation device. Therefore, once put in acapsule, it is allowed that the whole or part of particles are liberatedto separate particles by external agitation and thus forming a powderwithin the capsule.

BRIEF DESCRIPTION OF THE FIGURES

[0031]FIG. 1 is a graph illustrating the effect of nonionic surfactants.

[0032]FIG. 2 is a graph illustrating the effect of water-soluble,nonionic, organic binders.

[0033]FIG. 3 is a graph illustrating the effect of hydrogenatedlecithin.

[0034]FIG. 4 shows blood concentration profiles of human growth hormonein rat after transpulmonary administration of a human growthhormone-containing powder and subcutaneous injection of the same amountof the powder.

DETAILED DESCRIPTION OF THE INVENTION

[0035] In the present invention, the term an “aqueous liquid containinga physiologically active peptide” includes not only a simple aqueoussolution of a physiologically active peptide but also a solution of aphysiologically active peptide further containing one or more othercomponents that do not adversely affect the stability of thephysiologically active peptide, e.g., buffering agents such asphosphates, pharmaceutically acceptable salts such as sodium chloride,and diluents such as sorbitol.

[0036] As the method for stabilization of the present inventionstabilizes a physiologically active peptide dissolved in an aqueousliquid in a process of evaporating water from the aqueous liquid, itsstabilization effect on a physiologically active peptide is not affectedeven by drying the physiologically active peptide coated on the surfaceof larger particles chemically inert to the active peptide, such aslactose and the like. Such inert particles serve as cores which carry ontheir surface a coat of the physiologically active peptide mixed withone or more stabilizing agents

[0037] The powder of the present invention containing a physiologicallyactive peptide is based on the discovery that a very efficienttranspulmonary absorption is attained by employing particles comprisinga physiologically active peptide and mannitol. Thus, any method ofpreparation may be chosen as desired for preparing such powdercontaining a physiologically active peptide. The method of the presentinvention for preparation of a powder containing a physiologicallyactive peptide is based also on the discovery that mannitol has aneffect of remarkably stabilizing a physiologically active peptide in theprocess of forming a powder by drying an aqueous liquid containing thepeptide. Thus, drying of an aqueous solution containing aphysiologically active peptide and mannitol may be performed by anyconventional method as desired.

[0038] In the present invention, an example of particularly preferredphysiologically active peptides is human growth hormone. In the presentinvention, the term “human growth hormone” means not only 22K hGHextractable from the pituitary of a human, which consists 191 aminoacids and has a molecular weight of 22,125, but also 20K hGH, whichlacks 15 amino acids corresponding amino acids 32-46. 20K hGH has agrowth stimulating effect comparable to 22 K hGH. In the presentinvention, the term “human growth hormone” means not only these naturaltypes of human growth hormones, but also proteins which are produced byapplication of genetic recombination technology and having asubstantially comparable effect to the natural human growth hormones.Examples of human growth hormone produced by application of geneticrecombination technology include a N-terminal methionine-type hormoneconsisting 192 amino acids and variants which have part of their aminoacids deleted, substituted, added or inserted and having a comparableactivity to the natural types of human growth hormone.

EXAMPLES

[0039] When vigorously agitated in its aqueous solution, the molecule ofgrowth hormone (GH), among active peptides, readily undergoes alterationin tertiary structure in contact with a gas-liquid interface, resultingin the loss of its monomer and leading to the formation of its dimer,polymer or insoluble aggregates, as well as to the formation ofdeamidation products. As the gas-liquid interface is expanded in adrying process such as spray drying, it is necessary, particularly inthe case of GH, to manage to minimize its denaturation induced by thisexpansion of the interface in the process of forming GH into a powder.Studies were made as described below in search of a compound thatstabilizes GH. In the Examples and Control Examples below, human growthhormone was chosen as a representative of active peptides.

[0040] The human growth hormone employed in the Examples and ControlExamples below was a recombinant human growth hormone (in whichN-terminal methionine had been selectively deleted enzymaticaly) whichhad the same amino acid sequence as the natural human growth hormoneconsisting of 191 amino acids (22K hGH). In addition, where therecombinant human growth hormone is identified as “Growject Injection 4IU”, it indicates that a pharmaceutical product (Growject Injection 4IU: JCR pharmaceuticals, Co., Ltd.) was employed there. The compositionof Growject Injection 4 IU is as follows. Where the recombinant humangrowth hormone is specifically noted as a “bulk material”, it indicatesthe pure recombinant human growth hormone (produced by BTG), which isfree of any additives.

[0041] r-hGH Injection (Growject Injection 4 IU): (Formula) (Formula)r-hGH 4 IU (1.7 mg) Disodium hydrogenphosphate 2.2 mg Sodiumdihydrogenphosphate 0.35 mg Sodium chloride 1.0 mg D-mannitol 20.0 mg

[0042] Tests will be described below which were performed by spraydrying as a representative model of drying processes of an aqueousliquid containing a physiologically active peptide. The apparatusemployed for spray drying was Spray Dryer SD-1000 (EYELA).

[0043] As an indicator of stabilization of the physiologically activepeptide r-hGH, recovery rate of the physiologically active peptidemonomer was employed, for it is considered to be the best indicator ofstabilization of the physiologically active peptide. Calculation ofrecovery rate was done according to the following equation, based on theconcentration of the physiologically active peptide in the aqueousliquid before drying (before spray drying) and the content of recoveredactive peptide in the solution prepared by reconstituting the obtainedpowder (spray dried product) to the initial volume

[0044] Recovery rate of physiologically active peptide monomer(%)=A_(P)/A_(I)×100 where:

[0045] A_(P)=area of monomer peak on HPLC for spray dried product, and

[0046] A_(I)=area of monomer peak on HPLC before spray drying.

CONTROL EXAMPLE 1

[0047] To each of fifteen vials of the r-hGH injection (GrowjectInjection 4 IU) was added 1.0 ml of purified water to completelydissolve the injection. The r-hGH solution thus obtained (15 vials: 15.0ml) was spray-dried to obtain a dry powder. The conditions for spraydrying in the Spray Dryer SD-1000 were adjusted as follows.

[0048] Spray Drying Conditions

[0049] Inlet temperature: 80° C.

[0050] Atomizing pressure: 150 kPa

[0051] Dry air flow: 0.3 m³/min

[0052] Liquid feeder pump flow: 2.6 mL/min

[0053] The conditions for HPLC for determination of the monomer contentwere as follows.

[0054] HPLC Conditions

[0055] Apparatus: LC10A (SHIMADZU CORPORATION)

[0056] Detector: UV (280 nm)

[0057] Analyzing column: TSK G3000SW_(XL)

[0058] Column temperature: Room temperature

[0059] Mobile phase: 50 mM sodium dihydrogenphosphate, 50 mM disodiumhydrogenphosphate, 0.2 M sodium chloride.

[0060] Flow rate: 0.6 mL/min

[0061] Injection volume: 50 μL

CONTROL EXAMPLE 2

[0062] Five sets of r-hGH injection (Growject injection 4IU) vials, 15vials per set, were provided. To each of the vials was added 1.0 mL ofpurified water to completely dissolve the injection. The r-hGH solutionthus obtained (15.0 ml: 15 vials per set) was spray dried to obtain adry powder. The conditions for spray drying in the Spray Dryer SD-1000were different from those in Control Example 1 and adjusted as follows.The HPLC conditions for determination of the monomer content were thesame as those in Control Example 1.

[0063] Spray Drying Conditions

[0064] Inlet temperature: 90° C.

[0065] Atomizing pressure: 100 kPa

[0066] Dry air flow: 0.2 m³/min

[0067] Fluid feeder pump flow: 2.6 mL/min

EXAMPLE 1

[0068] As solutions of a nonionic surfactant, aqueous solutionscontaining Tween 20 at different concentrations (concentration: 0.01,0.05, 0.1, 0.5, 1.0 and 2.0 w/w %) were prepared. Fifteen vials of ther-hGH injection (Growject Injection 4IU) were provided for each of theaqueous solutions containing Tween 20 at the different concentrations.The aqueous solutions containing Tween 20 at different concentrationswere added to corresponding 15 vials, 1.0 mL each, and the injection wascompletely dissolved. Thus obtained r-hGH solutions containing Tween 20at different concentrations (15.0 mL: 15 vials for each Tween 20concentration) were spray-dried to obtain dry powders. The conditionsfor spray drying and HPLC were the same as those in Control Example 1.

EXAMPLE 2

[0069] As solutions of a nonionic surfactant, aqueous solutionscontaining HCO-60 (polyoxyethylenehydrogenated castor oil) at differentconcentrations (concentration: 0.01, 0.05, 0.1, 0.5, 1.0 and 2.0 w/w %)were prepared. Fifteen vials of the r-hGH injection (Growject Injection4IU) were provided for each of the aqueous solutions containing HCO-60at different concentrations. The aqueous solutions containing HCO-60 atdifferent concentrations were added to corresponding 15 vials, 1.0 mLeach, and the injection was completely dissolved. Thus obtained r-hGHsolutions containing HCO-60 at different concentrations (15.0 mL: 15vials for each HCO-60 concentration) were spray-dried to obtain drypowders. The conditions for spray drying and HPLC were the same as thosein Control Example 1.

EXAMPLE 3

[0070] As solutions of a nonionic surfactant, aqueous solutionscontaining Pluronic F68 (polyoxyethylene(160)polyoxypropylene(30)glycol) at different concentrations (concentration: 0.01, 0.05, 0.1,0.5, 1.0 and 2.0 w/w %) were prepared. Fifteen vials of the r-hGHinjection (Growject Injection 4IU) were provided for each of the aqueoussolutions containing Pluronic F68 at different concentrations. Theaqueous solutions containing Pluronic F68 at different concentrationswere added to corresponding 15 vials, 1.0 mL each, and the injection wascompletely dissolved. Thus obtained r-hGH solutions containing PluronicF68 at different concentrations (15.0 mL: 15 vials for each Pluronic F68concentration) were spray-dried to obtain dry powders. The conditionsfor spray drying and HPLC were the same as those in Control Example 1.

EXAMPLE 4

[0071] As solutions of a water soluble, nonionic, organic binder,aqueous solutions containing Kollidone 17PF (polyvinylpyrrolidone: BASF)at different concentrations (concentration: 0.01, 0.05, 0.1, 0.5, 1.0and 2.0 w/w %) were prepared. Fifteen vials of the r-hGH injection(Growject Injection 4IU) were provided for each of the aqueous solutionscontaining Kollidone 17PF at different concentrations. The aqueoussolutions containing Kollidone 17PF at different concentrations wereadded to corresponding 15 vials, 1.0 mL each, and the injection wascompletely dissolved. Thus obtained r-hGH solutions containing Kollidone17PF at different concentrations (15.0 mL: 15 vials for each Kollidone17PF concentration) were spray dried to obtain dry powders. Theconditions for spray drying and HPLC were the same as those in ControlExample 1.

EXAMPLE 5

[0072] As a water soluble, nonionic, organic binder, aqueous solutionscontaining Kollidone 12PF (polyvinylpyrrolidone: BASF) at differentconcentrations (concentration: 0.01, 0.05, 0.1, 0.5, 1.0 and 2.0 w/w %)were prepared. Fifteen vials of the r-hGH injection (Growject Injection4IU) were provided for each of the aqueous solutions containingKollidone 12PF at different concentrations. The aqueous solutionscontaining Kollidone 12PF at different concentrations were added tocorresponding 15 vials, 1.0 mL each, and the injection was completelydissolved. Thus obtained r-hGH solutions containing Kollidone 12PF atdifferent concentrations (15.0 mL: 15 vials for each Kollidone 12PFconcentration) were spray-dried to obtain dry powders. The conditionsfor spray drying and HPLC were the same as those in Control Example 1.

EXAMPLE 6

[0073] As a water soluble, nonionic, organic binder, aqueous solutionscontaining HPC-SSL (hydroxypropylcellulose: TOSOH) at differentconcentrations (concentration: 0.01, 0.05, 0.1, 0.5 and 1.0 w/w %) wereprepared. Fifteen vials of the r-hGH injection (Growject Injection 4IU)were provided for each of the aqueous solutions containing HPC-SSL atdifferent concentrations. The aqueous solutions containing HPC-SSL atdifferent concentrations were added to corresponding 15 vials, 1.0 mLeach, and the injection was completely dissolved. Thus obtained r-hGHsolutions containing HPC-SSL at different concentrations (15.0 mL: 15vials for each HPC-SSL concentration) were spray-dried to obtain drypowders. The conditions for spray drying and HPLC were the same as thosein Control Example 1.

EXAMPLE 7

[0074] As solutions of a nonionic surfactant, aqueous solutionscontaining Lecinol S-10E (hydrogenated lecithin: NIKKO CHEMICALS) atdifferent concentrations (concentration: 0.01, 0.05, 0.1, 0.5, 1.0 and2.0 w/w %) were prepared. Fifteen vials of the r-hGH injection (GrowjectInjection 4IU) were provided for each of the aqueous solutionscontaining hydrogenated lecithin at different concentrations. Theaqueous solutions containing hydrogenated lecithin at differentconcentrations were added to corresponding 15 vials, 1.0 mL each, andthe injection was completely dissolved. Thus obtained r-hGH solutionscontaining hydrogenated lecithin at different concentrations (15.0 mL:15 vials for each hydrogenated lecithin concentration) were spray-driedto obtain dry powders. The conditions for spray drying and HPLC were thesame as those in Control Example 1.

EXAMPLE 8

[0075] Aqueous solutions were prepared which contained HPC-SSL(hydroxypropylcellulose) and a nonionic surfactant in combination asindicated in the following table. TABLE 1 Aqueous Concentration ofNonionic surfactant and solution No. HPC-SSL (w/w %) its concentration(w/w %) A 0.05 HCO-60 0.05 B 0.05 Pluronic F68 0.05 C 0.05 Pluronic F680.10 D 0.10 HCO-60 0.05 E 0.10 HCO-60 0.10

[0076] Fifteen vials of the r-hGH injection (Growject Injection 4IU)were provided for each of the aqueous solutions containing HPC-SSL and anonionic surfactant in different combinations. The aqueous solutionswere added to a corresponding set of 15 vials, 1.0 mL each, and theinjection was completely dissolved. Thus obtained r-hGH solutions (15.0mL: per set of 15 vials for each combination) were spray-dried to obtaindry powders. The conditions for spray drying were the same as in ControlExample 2, and HPLC conditions were the same as those in Control Example1.

[0077] Results of Analysis

[0078]FIG. 1 shows the results of HPLC analysis performed in ControlExample 1 and Examples 1-3.

[0079] As shown in the figure, the nonionic surfactants atconcentrations in certain ranges, respectively, remarkably increased therecovery rate of the monomer of physiologically active peptide r-hGH inthe process of powder preparation from its aqueous solutions. While thecontent of r-hGH monomer fell to the order of 40% during powderpreparation in Control Example 1, which employed no nonionic surfactant,r-hGH monomer was maintained in Examples 1-3, in contrast at much higherrecovery rate in the samples containing 0.01-0.5 w/w % nonionicsurfactants in aqueous solutions. The figure also shows that thestabilizing effect of the respective nonionic surfactants peaked attheir concentrations of somewhere around 0.1 w/w %. Though lackingactually measured values, it is also evident, for example, that thenonionic surfactants at about 0.3 w/w % have higher stabilizing effectsthan at 0.5 w/w %.

[0080]FIG. 2 shows the results of HPLC analysis performed in ControlExample 1 and Examples 4-6.

[0081] As shown in the figure, the water-soluble, nonionic, organicbinders markedly increased the recovery rate of the monomer ofphysiologically active peptide r-hGH in the process of powderpreparation from its aqueous solutions. In Examples 4 (Kollidone 17PF)and 5 (Kollidone 12PF), improvement was noted at any of theirconcentrations tested. Their stabilizing effect was particularly potentup to a concentration of 1 w/w % and peaked at a concentration of 0.1w/w %. As for Example 6 (hydroxypropylcellulose), stabilizing effect wasstill more remarkable than where the other binders were employed,showing a r-hGH recovery rate of about 95% at a concentration of 0.1 w/w%, where its effect peaked. In Example 6, hydroxypropylcellulose wastested only up to the concentration of 1 w/w %. However, it is largelyevident that hydroxypropylcellulose would show a stabilizing effect evenat 2 w/w %. This is because its effect at 1 w/w % was much higher thanthe effects of the other organic binders employed in Example 4 and 5 atthe same concentration, and the decline in its effect by increasing itsconcentration beyond the peak is substantially not greater than thedecline seen in the graphs for Examples 4 or 5.

[0082]FIG. 3 shows the results of HPLC analysis performed in ControlExample 1 and Example 7.

[0083] As shown in the figure, hydrogenated lecithin, which was employedin Example 7, exhibited a remarkably potent stabilizing effect on r-hGHin any of the tested concentrations up to 2 w/w %. In particular, evenat 0.01 w/w %, i.e. the lowest concentration tested, hydrogenatedlecithin exhibited a stabilizing effect, raising the r-hGH recovery rateto more than 70%. Beyond that concentration and up to 0.2 w/w %,hydrogenated lecithin exhibited still higher stabilizing effects. Whileit seems from the figure that the effect of hydrogenated lecithin peaksat a concentration somewhere around 0.5-1 w/w %, its effect declinesonly slightly by increasing concentration beyond its peak. Therefore,there is no doubt that hydrogenated lecithin has a remarkablestabilizing effect in a concentration range much wider than testedabove.

[0084] Following Table 2 shows the results of HPLC analysis forComparison Example 2 and Example 8. TABLE 2 Concentration Nonionicsurfactant Aqueous of HPC-SSL and its Monomer solution No. (w/w %)concentration (w/w %) recovery rate (%) Control — — 64.04 ± 1.30 Example2 Example 8 A 0.05 HCO-60 0.05 99.20 ± 1.16 Example 8 B 0.05 PluronicF68 0.05 98.42 ± 0.61 Example 8 C 0.05 Pluronic F68 0.10 97.96 ± 1.34Example 8 D 0.10 HCO-60 0.05 104.76 ± 0.68  Example 8 E 0.10 HCO-60 0.10104.81 ± 0.17 

[0085] As seen in Table 2, r-hGH was stabilized substantially perfectlyin the process of forming its aqueous solution into a powder, byaddition of both of hydroxypropylcellulose and a nonionic surfactant tothe aqueous solution. This indicates that a combined use of both thewater-soluble, nonionic organic binder—hydroxypropylcellulose—and anonionic surfactant provides a higher stabilizing effect than by usingthem separately.

[0086] As seen from the results in Control Examples 1 and 2 and Examples1-8, stability of physiologically active peptide r-hGH in a process offorming a powder from the aqueous solution of the physiologically activepeptide is remarkably improved by adding to the solution; a nonionicsurfactant such as polysorbate, polyoxyethylenehydrogenated castor oiland poloxamer and the like; a water-soluble, nonionic, organic bindersuch as hydroxypropylcellulose and polyvinylpyrrolidone and the like; orhydrogenated lecithin. Moreover, addition of two or more of thesecomponents in combination further improves the stability of thephysiologically active peptide, leading to almost completestabilization.

EXAMPLE 9

[0087] Further studies were performed on the effect of mannitol, eitheremployed alone or in combination with other additives.

[0088] Materials

[0089] As GH, a recombinant human growth hormone (r-hGH) bulk materialwas used. As stabilizers, D-mannitol, HPC-SSL and Pluronic F68 wereused.

[0090] Preparation of r-hGH Solution

[0091] According to the following formulas, r-hGH and additives wereweighed and dissolved in 15.0 mL of purified water to prepare spraysolutions. As a control, r-hGH alone was dissolved in 15.0 mL ofpurified water to prepare a spray solution (Control Formula). In theformulas, “% by weight” in parentheses indicates the ratio of the weightof respective solid component to the weight of the solid components as awhole. (Formula M) r-hGH 29.25 mg (6.5% by weight) D-mannitol 420.75 mg(93.5% by weight) Total 450.00 mg (Formula M-HP) r-hGH 29.25 mg (6.5% byweight) D-mannitol 405.00 mg (90.0% by weight) HPC-SSL 15.75 mg (3.5% byweight) Total 450.00 mg (Formula M-P) r-hGH 29.25 mg (6.5% by weight)D-mannitol 405.00 mg (90.0% by weight) Pluronic F68 15.75 mg (3.5% byweight) Total 450.00 mg

[0092] Spray Drying

[0093] As a spray dryer, EYELA SD- 1000 Spray Dryer were used. Drypowders were prepared by spray-drying the above r-hGH solutions. Theconditions for spray drying was as follows.

[0094] Inlet temperature: 90° C.

[0095] Dry air flow: 0.2 m³/min

[0096] Atomizing pressure: 100 kPa

[0097] Fluid feeder pump flow: 2.6 mL/min

[0098] HPLC/Monomer Content Determination

[0099] The conditions for HPLC for determination of r-hGH monomer wereas follows.

[0100] Apparatus: LC10A (SHIMADZU CORPORATION)

[0101] Sample amount: about 0.02 g/0.5 mL purified water

[0102] Detector: UV (280 nm)

[0103] Analyzing column: TSK G3000SW_(XL) (TOSOH)

[0104] Column temperature: Room temperature

[0105] Mobile phase: 0.1 M sodium dihydrogenphosphate, 0.1 M disodiumhydrogenphosphate, 0.2 M sodium chloride.

[0106] Flowrate: 0.6 mL/min

[0107] Injection volume: 50 μL

[0108] HPLC/Determination of the Content of Deamidation Product

[0109] The conditions for HPLC for determination of r-hGH deamidationproducts were as follows.

[0110] Apparatus: LC10A (SHIMADZU CORPORATION)

[0111] Sample amount: about 0.02 g/0.5 mL purified water

[0112] Detector: UV (280 nm)

[0113] Analyzing column: Protein C4 column (VYDAC, Cat. No. 214ATP54)

[0114] Column temperature: 45° C.

[0115] Mobile phase: 50 mM Tris-HCl (pH 7.5)/n-propanol (71:29) buffer

[0116] Flow rate: 0.5 mL/min

[0117] Injection volume: 50 μL

[0118] SDS-polyacrylamide Gel Electrophoresis

[0119] 1) Preparation of Samples

[0120] Solutions of about 0.04 mg/mL was prepared as samples. To each 10μL of the solutions was added 10 μL of water and 20 μL of the samplebuffer. As a standard sample, a solution of about 1.6 mg r-hGH bulkmaterial/mL was prepared, to 10 μL of which was added 10 μL of water and20 μL of the sample buffer.

[0121] 2) Preparation of Electrophoresis Buffer

[0122] (A) An electrophoresis buffer for 10×SDS-PAGE was prepared byadding water to 30.3 g of Tris, 144 g of glycine and 10 g of SDS to makeinto volume of 1000 mL (for stock).

[0123] (B) An electrophoresis buffer for SDS-PAGE was prepared by adding900 mL of water to 100 mL of the electrophoresis buffer for 10×SDS-PAGE.

[0124] (C) A 0.25 M Tris-HCl buffer (pH 6.8) was prepared by addingwater to 30.25 g of Tris to make into volume of 800 mL, then adjustingthe pH of the solution to 6.8 with 6 N hydrochloric acid, and makinginto volume of 1000 mL with water (preserved by freezing).

[0125] (D) A sample buffer for SDS-PAGE was prepared by adding water to25 mL of 0.25 M Tris-HCl buffer (pH 6.8), 2 g of SDS, 5 g of sucrose and2 mg of bromphenol blue (BPB) to make 50 mL.

[0126] 3) SDS-PAGE

[0127] Using the samples and the buffer described above, electrophoresiswas carried out in a conventional manner at 20 mA/gel.

[0128] Results

[0129] The table below shows the results of the determination of thecontents of r-hGH monomer and deamidation products in the r-hGH powdersprepared above by freeze drying. TABLE 3 Content of Deamidation Formular-hGH Recovery Rate (%) Products (%) Control 68.5 7.3 M 80.5 4.2 M-HP91.4 4.5 M-P 88.4 4.7 Bulk Material — 3.1

[0130] As evident from the Table 3, r-hGH monomer recovery rate was muchhigher in any of Formulas M, M-HP, M-P than in the control formula:while the recovery rate of r-hGH in the control formula was 68.5%, thatwas 80.5% in Formula M. In Formulas M-HP and M-P, r-hGH recovery ratewas still higher. The content of deamidation products in any of theFormulas M, M-HP and M-P, which was lower than that in the controlformula, was substantially not different from the proportion (3.1%) ofdeamidation products contained originally in the bulk material employed.In the control formula, in contrast, the content of deamidation productsincreased beyond two times. The analysis by SDS-PAGE also showedelectrophoretic patters indicating that the purity of the peptide washigher in Formula M than in the control formula, and that the purity inFormula M-HP and M-P, in turn, was still higher than that in Formula M.

EXAMPLE 10 Mannitol-containing r-hGH Powder for TranspulmonaryAdministration for In Vivo Test

[0131] According to the following formula, r-hGH, HPC-SSL and D-mannitolwere weighed and dissolved in 90 mL of purified water to obtain a spraysolution. In the formula, “% by weight” in parentheses indicates theratio of the weight of respective solid component to the weight of thesolid components as a whole. In the spray solution, the concentration ofr-hGH, HPC-SSL and D-mannitol is 0.27% by weight, 0.14% by weight and2.92% by weight, respectively. Spray drying and analyses were performedunder the same conditions as in Example 9. (Formula) r-hGH 0.240 g (8.0%by weight) HPC-SSL 0.129 g (4.3% by weight) D-mannitol 2.631 g (87.7% byweight) Total 3.000 g

[0132] The r-hGH dry powder prepared by spray drying was observed byoptical microscopy. Six hundred particles were randomly chosen tomeasure their particle size. As a result, the particle size was found tobe 2.84±0.83 μm (mean ±SD, n=600). The areas of the main peak (%) andthe peak (%) for deamidation products were as follows. TABLE 4 Area ofMonomer Area of Deamidation Products Peak (%) Peak (%) Spray-dried 95.54.5 Product Standard 96.6 3.4

[0133] Pharmacokinetic Evaluation of GH Powder after TranspulmonaryAdministration to Rats

[0134] The GH powder was administered to rats for pharmacokineticevaluation. The same amount of the GH powder as transpulmonarilyadministered was dissolved in water and subcutaneously administered torats to compare its pharmacokinetics with that of transpulmonarilyadministered GH.

[0135] Test Animals

[0136] Six male 9-week-old Wistar rats were used for transpulmonary andsubcutaneous injection, respectively.

[0137] GH Powder Tested

[0138] The r-hGH powder obtained in Example 10 above was used.

[0139] Administration of r-hGH

[0140] After fasting for a full day and night, the rats of thetranspulmonary administration group were anesthetized with urethane. Twomg/kg rat body weight of the r-hGH powder was placed in a transpulmonaryadministration device for rats (PennCentury). The powder was dischargedinto the lungs of the rats through the device's delivery tube insertedin the trachea by thrusting out 3 mL of air from a syringe connected tothe device. The rats of the subcutaneous administration group were alsofasted for a full day and night and then subcutaneously injected withthe r-hGH powder suspended in purified water in an amount equivalent to2 mg/kg rat body weight.

[0141] Blood Sampling and Processing

[0142] Blood sampling was performed just before the administration ofr-hGH and then 0, 15, 30, 60, 120, 240, 480 and 1440 minutes thereafter.Blood was sampled from the cervical vein of restrained rats. Bloodsampling volume was 300 μL at one time. Following each blood sampling,the same amount (300 μL) of physiological saline was injected into thecervical vein. Blood samples were let stand for one hour at roomtemperature and then overnight at 4° C., and centrifuged (15,000 rpm, 10minutes, 4° C.) to separate the sera.

[0143] Measurement of Blood r-hGH Concentration by GH-ELISA

[0144] An anti-hGH rabbit polyclonal antibody raised by a conventionalmethod was diluted and adjusted to the absorbance OD280 of 0.02 with a0.05 M Tris buffer. The solution was placed, 100 μL each, in the wellsof 96-well plates and incubated for two hours at 37° C. The plates werewashed five times with a 0.01 M phosphate buffer (washing buffer). Thewells of the plates were filled with a block solution (Block Ace:Dainippon Pharmaceutical Co., Ltd.) and let stand overnight at 4° C. Thesera obtained above and r-hGH for a standard curve, respectively, werediluted as needed with 10×Block Ace aqueous solution and added to thewells of the washed plates, 100 μL each, and preincubated for 2 hours at37° C.

[0145] Using the anti-hGH rabbit polyclonal antibody, a horseradishperoxidase (HRP)-conjugated anti-hGH rabbit polyclonal antibody wasprepared in a conventional manner. The conjugated polyclonal antibodywas diluted 50,000 times with 10×Block Ace aqueous solution and added tothe wells of the washed plates, 100 μL each, and preincubated for 2hours at 37° C. After washing, 100 μL each of TMB reagent (BIORAD) wasadded to the wells of the plates and allowed to react for 10 minutes atroom temperature. The reaction was terminated by addition of 1 Nsulfuric acid, and absorbance was measured at 450 nm. A calibrationcurve was created based on the absorbance for standard solutions, andthe r-hGH concentrations in the samples were derived from theirabsorbance using the calibrative curve.

[0146] Results

[0147] Table 5 and FIG. 4 show r-hGH concentrations in the blood aftertranspulmonary administration of the r-hGH powder or subcutaneousinjection of the r-hGH suspension. TABLE 5 Time after Blood r-hGHConcentration (ng/ml) Administration Transpulmonary Subcutaneous (min)Administration Injection 0 22.6 41.5 15 584.4 423.1 30 451.4 446.1 60315.1 491.8 120 254.9 423.9 240 101.6 347.9 480 61.5 175.5 1440 34.751.3

[0148] As seen in Table 5 and FIG. 4, after transpulmonaryadministration of the r-hGH powder prepared in the above example, bloodr-hGH concentration reached its peak of 584.4 ng/mL 15 minutes after theadministration. The concentration then started to decline but stillremained at 34.7 ng/mL even 1440 minutes after administration. The AUC(area under the curve representing blood pharmacokinetics) up to 480minutes after the administration was 128862 ng/mL·min for transpulmonaryadministration, whereas that was 255826 ng/mL·min for subcutaneousadministration of the suspension containing the same amount of thepowder. As the r-hGH was transferred to the blood in unexpectedly highefficiency after the subcutaneous injection of that composition, theblood concentration of r-hGH following transpulmonary administration waslower than that following its subcutaneous injection, except for aperiod immediately after pulmonary administration. However, the aboveresults show that r-hGH absorption after transpulmonary administrationof the composition was very high. In fact, the blood r-hGH concentrationafter the transpulmonary administration of the very composition was farhigher than either of the blood r-hGH concentration after thetranspulmonary administration or subcutaneous administration of the sameamount of r-hGH suspension carried out in Control Example 3 below.

CONTROL EXAMPLE 3

[0149] According to the following formula, r-hGH and lactose wereweighed and dissolved in 120 mL of purified water to obtain a spraysolution. The concentration of r-hGH and lactose in the spray solutionis 0.20 w/w % and 2.30 w/w %, respectively.

[0150] Formula

[0151] r-hGH 0.240 g (8.0% by weight)

[0152] Lactose (monohydrate) 2.760 g (92.0% by weight)

[0153] Total 3.000 g

[0154] The above spray solution was spray-dried under the followingconditions.

[0155] Inlet temperature: 120° C.

[0156] Dry air flow: 0.2 m³/min

[0157] Atomizing pressure: 100 kPa

[0158] Fluid feeder pump flow: 2.6 mL/min

[0159] Thus obtained stray-dried r-hGH powder was analyzed by the samemethod as described above for the content of monomer and deamidationproducts by HPLC, and subjected to SDS-PAGE. The area of the monomerpeak and that of the peak of deamidation products are as shown in thefollowing table, and the SDS-PAGE pattern indicated high purity. TABLE 6Area for Deamidation Area for Monomer (%) Products (%) Spray-dried 94.75.3 Product Standard 96.7 3.3

[0160] With this spray-dried product, r-hGH was transpulmonarilyadministered or subcutaneously injected to male 9-week-old Wistar rats,six animals per group, following the same dose and procedures asindicated in “Pharmacokinetic Evaluation of GH Powder afterTranspulmonary Administration to Rats”, and the pharmacokinetics forr-hGH was determined. The results are shown in the table below. TABLE 7Time after Blood r-hGH Concentration (ng/ml) AdministrationTranspulmonary Subcutaneous (min) Administration Injection 0 5 6 15 14746 30 129 52 60 85 62 120 70 75 240 58 71 480 48 21 1440 37 5

[0161] Natural human growth hormone, 22K hGH, is composed of 191 aminoacids, with two S-S bonds within the molecule, whereas human insulin iscomposed of 51 amino acids and has two S-S bonds within the molecule. Itis reasonably expected that transpulmonary absorption demonstrated abovewith the powders containing human growth hormone will occur also withhuman insulin, considering that the far smaller molecule of humaninsulin compared with human growth hormone will render the former moresuitable for absorption through mucous membranes and that it shares astructural similarity with human growth hormone in light that they havetwo S-S bonds within their molecule. Likewise, successful transpulmonaryabsorption is expected to take place also with calcitonin (32 aminoacids) and somatostatin (28 amino acids), which are roughly of half thesize of human insulin, by forming them into the powder of the presentinvention.

[0162] The present invention enables to remarkably stabilize aphysiologically active peptide in forming a powder by drying an aqueoussolution containing the physiologically active peptide, therebyminimizing loss of the peptide in the process of powder formation. As itis done employing additives approved as safety ingredients inpharmaceutical products, the present invention also enables to produce apowder stably retaining a physiologically active peptide, withoutevoking unnecessary concerns on the safety of such a product due toemployed additives. The present invention alto enables to providephysiologically active peptide-containing powder in which content ofdimers or other denatured peptide is minimized, thereby making it easyto produce such types of pharmaceutical compositions that are adapted tobe applied to mucous membranes in a powder form in order to introduce adrug into the circulating blood, e.g. pharmaceutical compositions fortransnasal or transpulmonary administration. The present inventionfurther enables to provide inhalant compositions which allow efficienttransferal of growth hormone or insulin into the blood by transpulmonaryadministration.

What is claimed is:
 1. A method for stabilization of a physiologicallyactive peptide in a process of preparing a powder containing thephysiologically active peptide by drying an aqueous liquid containingthe physiologically active peptide, wherein the method comprises addingto the aqueous liquid at least one compound selected from the groupconsisting of a nonionic surfactant, a water-soluble, nonionic, organicbinder, hydrogenated lecithin, and mannitol.
 2. A method forstabilization of a physiologically active peptide in a process ofpreparing a powder containing the physiologically active peptide bydrying an aqueous liquid containing the physiologically active peptide,wherein the method comprises adding to the aqueous liquid mannitol andat least one compound selected from the group consisting of a nonionicsurfactant, a water-soluble, nonionic, organic binder, and hydrogenatedlecithin.
 3. A method for stabilization of a physiologically activepeptide in a process of preparing a powder containing thephysiologically active peptide by drying an aqueous liquid containingthe physiologically active peptide, wherein the method comprises addingto the aqueous liquid at least one component selected from the groupconsisting of a nonionic surfactant in an amount of 0.01-0.5% by weight,a water-soluble, nonionic, organic binder in an amount of 0.01-1% byweight, hydrogenated lecithin, and 1-50 parts by weight of mannitol perone part by weight of the physiologically active peptide.
 4. A methodfor stabilization of a physiologically active peptide in a process ofpreparing a powder containing the physiologically active peptide bydrying an aqueous liquid containing the physiologically active peptide,wherein the method comprises adding to the aqueous liquid 1-50 parts byweight of mannitol per one part by weight of the physiologically activepeptide and at least one component selected from the group consisting ofa nonionic surfactant in an amount of 0.01-0.5% by weight, awater-soluble, nonionic, organic binder in an amount of 0.01-1% byweight, and hydrogenated lecithin.
 5. The method of one of claims 1 to 4wherein the water-soluble, nonionic, organic binder is selected from thegroup consisting of polyvinylpyrrolidone, a water-soluble, nonioniccellulose derivative, and polyvinylalcohol.
 6. The method of claim 5wherein the water-soluble, nonionic cellulose derivative is selectedfrom the group consisting of hydroxypropylcellulose,hydroxyethylcellulose, and hydroxypropylmethylcellulose.
 7. The methodof one of claims 1 to 6 wherein the nonionic surfactant is selected fromthe group consisting of polysorbate, polyoxyethylenehydrogenated castoroil, and a poloxamer.
 8. The method of one of claims 1 to 7 whereindrying of the aqueous liquid is performed by spray drying,lyophilization or spray-freeze drying, or by coating which may befluid-bed coating, or performed in fluid-bed granulation.
 9. The methodof one of claims 1 to 8 wherein the average size of the particles makingup the powder is 1-10 μm.
 10. The method of one of claims 1 to 9 whereinthe physiologically active peptide is selected from the group consistingof growth hormones, insulins, calcitonins, erythropoietin, glucagon,somatostatin, somatostatin derivatives, interferons, interleukins,superoxide dismutase, urokinase, proteases, tumor necrosis factors,colony-stimulating factors, kallikrein, lysozyme, fibronectin,insulin-like growth factors, epidermal growth factor, fibroblast growthfactors, platelet-derived growth factor, nerve growth factor, hepatocytegrowth factor, vasculogenesis factors, and anti-vasculogenesis factors.11. The method of one of claims 1 to 9 wherein the physiologicallyactive peptide is human growth hormone or human insulin.
 12. The methodof one of claims 1 to 9 wherein the physiologically active peptide ishuman growth hormone.
 13. A method for preparation of a powdercontaining a physiologically active peptide, wherein the methodcomprises forming a powder by drying an aqueous liquid containing aphysiologically active peptide and at least one compound selected fromthe group consisting of a nonionic surfactant, a water-soluble,nonionic, organic binder, hydrogenated lecithin, and mannitol.
 14. Amethod for preparation of a powder containing a physiologically activepeptide, wherein the method comprises forming a powder by drying anaqueous liquid containing the physiologically active peptide, mannitol,and at least one compound selected from the group consisting of anonionic surfactant, a water-soluble, nonionic, organic binder, andhydrogenated lecithin.
 15. A method for preparation of a powdercontaining a physiologically active peptide, wherein the methodcomprises forming a powder by drying an aqueous liquid containing thephysiologically active peptide and at least one component selected fromthe group consisting of a nonionic surfactant in an amount of 0.01-0.5%by weight, a water-soluble, nonionic, organic binder in an amount of0.01-1% by weight, hydrogenated lecithin and 1-50 parts by weight ofmannitol per one part by weight of the physiologically active peptide.16. A method for preparation of a powder containing a physiologicallyactive peptide, wherein the method comprises forming a powder by dryingan aqueous liquid containing the physiologically active peptide, 1-50parts by weight of mannitol per one part by weight of thephysiologically active peptide, and at least one component selected fromthe group consisting of a nonionic surfactant in an amount of 0.0 1-0.5%by weight, a water-soluble, nonionic, organic binder in an amount of0.01-1% by weight, and hydrogenated lecithin.
 17. The method forpreparation of a powder containing a physiologically active peptide ofone of claims 13 to 16, wherein the water-soluble, nonionic, organicbinder is selected from the group consisting of polyvinylpyrrolidone, awater-soluble, nonionic cellulose derivative, and polyvinylalcohol. 18.The method for preparation of a powder containing a physiologicallyactive peptide of claim 17, wherein the water-soluble, nonioniccellulose derivative is selected from the group consisting ofhydroxypropylcellulose, hydroxyethylcellulose, andhydroxypropylmethylcellulose.
 19. The method for preparation of a powdercontaining a physiologically active peptide of one of claims 13 to 18,wherein the nonionic surfactant is selected from the group consisting ofpolysorbate, polyoxyethylenehydrogenated castor oil, and a poloxamer.20. The method for preparation of a powder containing a physiologicallyactive peptide of one of claims 13 to 19, wherein drying of the aqueousliquid is performed by spray drying, lyophilization or spray-freezedrying, or by coating which may be fluid-bed coating, or performed influid-bed granulation.
 21. The method for preparation of a powdercontaining a physiologically active peptide of one of claims 13 to 20wherein the average size of the particles making up the powder is 1-10μm.
 22. The method for preparation of a powder containing aphysiologically active peptide of one of claims 13 to 21, wherein thephysiologically active peptide is selected from the group consisting ofgrowth hormones, insulins, calcitonins, erythropoietin, glucagon,somatostatin, somatostatin derivatives, interferons, interleukins,superoxide dismutase, urokinase, proteases, tumor necrosis factors,colony-stimulating factors, kallikrein, lysozyme, fibronectin,insulin-like growth factors, epidermal growth factor, fibroblast growthfactors, platelet-derived growth factor, nerve growth factor, hepatocytegrowth factor, vasculogenesis factors, and anti-vasculogenesis factors.23. The method for preparation of a powder containing a physiologicallyactive peptide of one of claims 13 to 21, wherein the physiologicallyactive peptide is human growth hormone or human insulin.
 24. The methodfor preparation of a powder containing a physiologically active peptideof one of claims 13 to 21, wherein the physiologically active peptide ishuman growth hormone.
 25. A powder containing a physiologically activepeptide, wherein the powder is made up of particles comprising aphysiologically active peptide and mannitol at a weight proportion of1:1 to 1:50.
 26. The powder containing a physiologically active peptideof claim 25, wherein the particles further comprise per one part byweight of the physiologically active peptide at least one componentselected from the group consisting of a nonionic surfactant in an amountof 0.05-3 parts by weight, a water-soluble, nonionic, organic binder inan amount of 0.05-6 parts by weight, and hydrogenated lecithin.
 27. Thepowder containing a physiologically active peptide of claim 25 or 26,wherein the average size of the particles is 1-10 μm.
 28. The powdercontaining a physiologically active peptide of one of claims 25 to 27,for which drying of the aqueous solution was performed by spray drying,spray-freeze drying, or lyophilization.
 29. The powder containing aphysiologically active peptide of one of claims 25 to 28, wherein thephysiologically active peptide is selected from the group consisting ofgrowth hormones, insulins, calcitonins, erythropoietin, glucagon,somatostatin, somatostatin derivatives, interferons, interleukins,superoxide dismutase, urokinase, proteases, tumor necrosis factors,colony-stimulating factors, kallikrein, lysozyme, fibronectin,insulin-like growth factors, epidermal growth factor, fibroblast growthfactors, platelet-derived growth factor, nerve growth factor, hepatocytegrowth factor, vasculogenesis factors, and anti-vasculogenesis factors.30. The powder containing a physiologically active peptide of one ofclaims 25 to 28, wherein the physiologically active peptide is humangrowth hormone or human insulin.
 31. The powder containing aphysiologically active peptide of one of claims 25 to 28, wherein thephysiologically active peptide is human growth hormone.
 32. An inhalantcomposition containing a physiologically active peptide, wherein theinhalant composition comprises particles as defined in one of claims 25to 31.